March 2009 Articles

Protein encounter complexes revealed

The PRE approach is very responsive to transient close approaches to protons of a usually distant paramagnetic spin label. This results from the steep distance dependence of the line broadening proportional to <r⁻⁶> over the range of the proton line broadening out to 24 Å or more. For protein-DNA and protein-protein associations driven by electrostatics, fitting of intermolecular PREs by rigid body docking visualized lightly populated (higher energy) encounter complexes differing from static structural coordinates [18,19]. This approach recently resolved the dilemma of how the apparently monomeric precursor to HIV protease activates itself into mature HIV protease dimer where the termini are sequestered in a β sheet remote from the active site [20]. Using a truncated precursor form, large intermolecular PREs were introduced to segments of the dimer interface by individual spin labels. The intermolecular PREs of the truncated precursor of HIV protease were modeled adequately by four-member structural ensembles in which dimers were populated to 3-5% with a similar interface with mature HIV protease but highly variable orientations [20] (Figure 2). Spin labeling near the N terminus suggests that, in a subset of the transient dimers, the N terminus makes transient contacts with the active site. This accounts for how the otherwise mostly monomeric precursor of HIV protease can process the N terminus to generate the mature full length [20].

Figure 2.

The predominantly monomeric precursor of HIV protease accompanied by lightly populated dimeric species

The blue lines represent some of the multitude of orientations in the transient encounter complexes that the second precursor subunit may adopt relative to the monomer plotted. The lines connect the Cα atoms of Gly27 of the active site and Ile62 on the distant side of the protomer. In the mature dimer, this vector runs horizontally in the middle on the right. The red coloring marks segments for which the proton NMR peaks are broadened by intermolecular paramagnetic relaxation enhancements from nitroxide labeling of half of the molecules in the samples of the truncated precursor. Termini are labeled.

Another association involving an encounter complex was characterized instead by NMR relaxation dispersion, which reports on equilibrium kinetics, thermodynamics, and indirectly upon structural change. Exchange within milliseconds between states in equilibrium can contribute line broadening (R_ex) that depends on RF pulsing rate ν_CPMG in CPMG experiments. R_ex as a function of ν_CPMG and magnetic field strength can be fitted to the rate of interconversion k_ex between ‘sites’ or states, the population of each state that gives their free energy differences, and the chemical shift difference Δω between sites (the difference in spectral peak positions) [21]. The phoshorylated kinase-inducible activation domain (pKID) of transcription factor CREB (cAMP response element binding) is intrinsically unfolded but is structured when bound to the KIX domain of CREB-binding protein. Titrations suggested this association to involve an encounter complex that is only partly structured and that makes transient hydrophobic contacts with KIX [22]. The coupled binding and folding processes include an intermediate in the equilibrium: free ↔ encounter ↔ intermediate ↔ bound [22]. The line broadening data were fitted well by a model with three sites. The chemical shift changes Δω suggested the equilibrium to be among encounter complex, intermediate, and bound state [22]. The two helices of pKID behaved a little differently, with αB featuring a dissociation that was fivefold faster. In regard to the intermediate, Δω values suggested that αA nearly reached the bound structure but αB less so [22].

Dynamic energy landscape of enzyme catalysis

Rate constants k_ex of equilibrium conformational fluctuations, from CPMG relaxation dispersion studies, correlated elegantly with enzymatic rate constants of cyclophilin A [23] and dihydrofolate reductase (DHFR) (both chemical transformation and product release) [24]. The insights became even more profound by recognizing the structural implications of Δω obtained from fitting ¹⁵N relaxation dispersions of the conformational fluctuations of intermediates of DHFR. The intermediates are binary or ternary complexes with cofactor, substrate, product, or analogue [24]. Δω of the fluctuations was consistently highly correlated with chemical shift changes δ accompanying the addition of a ligand to shift a binary complex to the next or previous ternary complex in the catalytic cycle. This provides strong evidence that the higher energy substate of each DHFR intermediate adopts a structure very similar to that of the dominant ground state of the next or previous intermediate in the catalytic cycle [24]. This demonstration of transient excited states (populated to a few percent) being linked to the adjacent intermediate by protein conformational fluctuations is a landmark in understanding enzyme guidance through the dynamic energy landscape of its catalytic cycle [25].

Structures of sparsely populated conformers

The concept of conformational selection evident in the molecular recognition by DHFR [24] was recently also articulated for ubiquitin's associations with other proteins [26]. The latter case depended upon access to the structures of sparsely populated higher energy states, achieved by innovative use of RDCs. RDCs introduce peak splittings to NMR peaks of solutions aligned very weakly, typically by liquid crystalline components. The RDCs report on the orientation, relative to the small overall alignment, of bond vectors joining J-coupled nuclei [27].

An ensemble of ubiquitin conformers was fitted to many sets of backbone RDCs (arising from independent means of alignment) using a new strategy of structural calculations [26]. The diversity of the ensemble was validated by its consistency with both the conformational restriction of bonds indicated by backbone order parameters derived from the RDCs and principal components analysis [26]. The diversity of the conformations in the ensemble is so great as to encompass all of the conformations ever reported for ubiquitin complexes [26]. The regions of greater mobility and greater spread of conformations correlate very well with the sites of contacts within ubiquitin complexes. These results suggested that all of these conformations are accessed transiently, within microseconds, as sparsely populated higher energy substates that can be selected by the binding partners of ubiquitin when they associate [26].

Kay's group has developed an independent strategy using RDCs that is also a technical tour de force. The solution structure was determined for one particular ‘invisible’ bound state of an SH3 domain populated to approximately 5% with phosphopeptide [28]. The excited state's backbone dihedral angles were estimated from measurements of nitrogen, proton, and carbon chemical shifts by means of relaxation dispersion methods [21,29-31]. Bond orientations in the excited state were estimated from RDCs and residual carbonyl chemical shift anisotropies introduced by weak alignment, each measured using clever new relaxation dispersion schemes [32-35].